Composition and Methods for Affecting Metallocorrinoid Uptake

ABSTRACT

The present invention is directed to compositions and methods for affecting metallocorrinoid uptake. The compositions and methods of the present invention are particularly useful in enhancing the uptake or availability of biologically active metallocorrinoids (e.g. cobalamin and its analogs). The present invention is particularly useful in the treatment or prevention of conditions that result from low expression or activity of proteins involved in the processing of metallocorrinoids, as well as in conditions which would benefit from enhanced uptake or availability of cobalamin or its biologically active analogs of cobalamin (e.g. cobalamin drug conjugates).

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/511,085, filed on Aug. 28, 2006, which is a continuation of U.S.application Ser. No. 10/761,870, filed Jan. 21, 2004 (Now abandoned),which is a continuation of U.S. application Ser. No. 09/864,747, filedon May 24, 2001 (Now U.S. Pat. No. 6,752,986, issued on Jun. 22, 2004).The entire teachings of the above applications are incorporated hereinby reference.

BACKGROUND OF THE MENTION

Metallocorrinoids are corrin rings with a metal-atom center, such as Co,Fe, Ni, or Mn. A corrin ring is four reduced pyrrole rings linkedtogether. A subclass of naturally occurring metallocorrinoids is knownas cobalamin, that is, a cobalt-centered corrin ring. Naturallyoccurring vitamin B₁₂, for example, is a cobalamin.

Vitamin B₁₂ compounds are known to have many biological functions. Theyare required by the enzyme methionine synthase, for example, which isinvolved in the production of DNA. Pregnant women need increased amountsof vitamin B₁₂ which is involved in the production of red blood cells.It is also believed that vitamin B₁₂ enhances the effects of othervitamins and nutrients in tissue repair. Lack of vitamin B₁₂ leads tomegaloblastic anemia (characterized by large and immature red bloodcells) and neuropathy in man with insidious onset of symptoms. Thesesymptoms include weakness, tiredness, breathlessness (dyspnea) onexertion, tingling and numbness (paresthesia), sore tongue (glossitis),loss of appetite and weight, loss of sense of taste and smell,impotence, psychiatric disturbances (such as irritability, memoryimpairment, mild depression, hallucinations) and sever anemia (which maylead to signs of cardiac dysfunction). Deficiency of vitamin B₁₂ leadsto defective DNA synthesis in cells; tissues most affected are thosewith the greatest rate of cell turnover, e.g. the haematopoietic system.In small children Cbl deficiency can result in developmental delay,hematological disorders, and neurological disorders. There may beirreversible damage to the nervous system with specific demyelination ofthe spinal cord.

Increased availability of vitamin B₁₂, on the other hand, appears tohave a very beneficial effect. Cbl analogs and cobalamin drug conjugateshave been shown to inhibit the growth of leukemia cells by possiblydeactivating methionine synthase, thus preventing DNA synthesis. Thecobalamins that are analogous to vitamin B₁₂ compounds would appear tobe potential therapeutic agents. These include hydroxocobalamin,cyanocobalamin, nitrocobalamin, mehtylcobalamin, and5′-deoxyadenocobalamin, as well as nitrosylcobalamin.

All forms of vitamin B₁₂ (adenosyl-, cyano-, hydroxo-, ormethylcobalamin) are bound by the transport proteins intrinsic factorand transcobalamin II, to be biologically active. Those transportproteins involved in the uptake of vitamin B₁₂ are referred to herein ascobalamin binding proteins. Specifically, gastrointestinal absorption ofvitamin B₁₂ relies upon the intrinsic factor-vitamin B₁₂ complex beingbound by the intrinsic factor receptors in the terminal ileum. Likewise,intravascular transport and subsequent cellular uptake of vitamin B₁₂throughout the body is dependent upon transcobalamin II and the cellmembrane transcobalamin II receptors, respectively. After thetranscobalamin II-vitamin B₁₂ complex has been internalized, thetransport protein undergoes lysozymal degradation, which releasesvitamin B₁₂ into the cytoplasm.

Cellular utilization of Cbl is preceded by two importantreceptor-mediated endocytic events. First, the dietary Cbl bound togastric intrinsic factor (IF), a 50-kDa glycoprotein, is transportedacross the absorptive enterocyte via an intrinsic factor-cobalaminreceptor that is expressed exclusively in the apical or the luminalmembranes. The plasma transport of cobalamin to tissues/cells appears tooccur via transporter transcobalamin II (TC II), by receptor-mediatedendocytosis via transcobalamin II-receptor (TC II-R). Intracellularlyreleased Cbl is then converted to its biologically active forms, (e.g.methyl-Cbl and 5′-deoxyadenosyl-Cbl) which are utilized by thecytoplasmic enzyme methionine synthase (MS) and mitochondrial enzymemethyl-malonyl-CoA mutase (MMCM), respectively. MS activity is requiredfor folate metabolism and DNA synthesis and presents a promising targetto block cell proliferation. TCII and serum Cbl levels are bothincreased in hepatocarcinomas and leukemias. TCII has been identified asan acute phase reactant in autoimmune disorders and infection. Severalstudies have shown that high levels of Cbl inhibited L1210, P388D1,CCRF-CEM, and NCTC929 cell proliferation. This is likely due to theactivation of an autoimmune response.

Recent studies have shown that TC II-R is expressed as a non-covalenthomodimer of molecular mass of 124 kDa in tissue plasma membranes ofhuman, rat, and rabbit. A comprehensive review of transcobalamin II, thetranscobalamin II receptor, and the uptake of vitamin B₁₂ is provided in“Transcobalamin II and Its Cell Surface Receptor Vitamins and Hormones”,Vitamins and Hormones, Vol. 59, pgs. 337-366 (2000) which isincorporated herein in its entirety by reference thereto. Plasmamembrane expression of TC II-R appears important for the tissue/cellularuptake of Cbl since its functional inactivation in vivo by itscirculatory antiserum results in intracellular deficiency of Cbl. Thisintracellular deficiency in Cbl results in the development of Cbldeficiency of the animal as a whole.

The utilization of vitamin B₁₂ as a delivery vehicle is known art. Theart describes an oral delivery system that delivers active substances(hormones, bio-active peptides or therapeutic agents) by binding theseagents to cobalamin or an analog thereof.

U.S. Pat. No. 5,936,082, which is hereby incorporated by reference inits entirety, for example, describes the therapeutic effectiveness ofvitamin B₁₂ based compounds. Nitrosylcobalamin (NO-Cbl), in particular,was evaluated for its chemotherapeutic effect. In five humanhematological and eight solid tumor cell lines, NO-Cbl exhibited anID₅₀that was 5-100 fold lower in tumor cell lines compared to benigncells (fibroblasts and endothelial cells). When oxidized from NO-Cbl,the NO free radical functions in a number of capacities. NO is involvedin vasodilation, and is known to contribute to increased oxidativestress, inhibition of cellular metabolism and induction of DNA damageleading to apoptosis and/or necrosis.

Radiolabelled vitamin B₁₂ analogs have also been described in the art asuseful in vivo imaging agents. For example, U.S. Pat. No. 6,096,290,which is hereby incorporated herein in its entirety by referencethereto, describes the use of radiolabelled vitamin B₁₂ analogs as invivo tumor imaging agents.

U.S. Pat. No. 6,183,723, which is also incorporated herein by referencein its entirety, describes certain other cobalamin-drug conjugates.

SUMMARY OF THE INVENTION

The multiple components of Cbl uptake, enzymes, co-factors, andtransport systems present several points of attack for the therapeuticdelivery of cobalamins. As is described herein, the interrelationship ofTCII-R and cytokines make this an attractive target for the therapeuticdelivery of biologically active metallocorrinoids. Cytokines, inparticular interferon β, are shown to enhance the uptake or activity ofbiologically active metallocorrinoids, including vitamin B₁₂ analogs,homologs, and derivatives.

Vitamin B₁₂ analogs can be synthesized in a number of ways. In additionto conjugation of the side chains of the corrin ring, conjugation to theCbl moiety can also be made, as can conjugation to the ribose moiety,phosphate moiety, and to the benzimidazole moiety. The conjugating agentand the drug to be conjugated depend upon the type of Cbl group that ismodified and the nature of the drug. One of skill in the art wouldunderstand how to adapt the conjugation method to the particular Cblgroup and drug to be coupled.

Preferred methods of attaching the drug to the Cbl molecule includeconjugation to Cbl via biotin. Biotin is conjugated to either thepropionamide or the acetamide side chains of the corrin ring of the Cblmolecule. The initial biotin-Cbl complex can be prepared according toPathre, et al. (Pathre, P. M., et al., “Synthesis of Cobalamin-Biotinconjugates that vary in the position in cobalamin coupling, Evaluationof cobalamin derivative binding to transcobalamn II,” incorporated byreference). Vitamin B₁₂ is commercially available in its most stableform as cyanocobalamin from Sigma Chemical (St. Louis, Mo.).

One may most easily obtain transcobalamin II in the following manner:transcobalamin II cDNA is available in the laboratories of Drs.Seetharam (Medical College of Wisconsin) and Rothenberg (VA-Hospital,New York) TC II cDNA can be expressed in a Baculovirus system to make alarge amount of functionally active TC II protein (see Quadors, E. V.,et al., Blood 81: 1239-1245, 1993). One of skill in the art would beable to reproduce the TC II cDNA. The antibodies to TCII-R were obtainedthrough the laboratory of Dr. Bellur Seetharam, Med. College of WI.

One way to make cobalamin drug conjugates is through geneticengineering. In this method, a DNA sequence encoding TC II and thepeptide drug may be expressed as one chimeric molecule. For example, itis possible to generate a chimeric construct using the full-length TC IIcDNA and the cDNA for a peptide drug (e.g. insulin). The chimericconstruct can then be expressed to produce a fusion protein consistingof the TC II-peptide drug. Following synthesis, the chimeric proteinshould be tested for both TC II activity and drug activity. Cobalamincan then be allowed to bind to this chimeric protein and used fortherapy.

The observation that a cytokine (i.e an interferon such as interferon-β)upregulates or enhances the activity of the TCII-R provides a basis fora number of embodiments of the present invention.

One embodiment of the present invention is a method for increasingcobalamin-binding protein activity in a subject in order to treat acondition favorably affected by an increase in said cobalamin-bindingactivity, said method compromising the step of administering to asubject in need of such treatment a cytokine in an amount effective toincrease cobalamin-binding activity in the subject. This method mayfurther include the step of administering a vitamin B₁₂ analog (whichmay be a naturally occurring vitamin B₁₂ analog), nitrosylcobalamin orother suitable vitamin B₁₂ drug conjugate. In this embodiment, thecytokine may be administered prior, simultaneously, or consecutivelywith the vitamin B₁₂ analog. The cytokine and/or vitamin B₁₂ analog maybe administered prophylactically or acutely. The increased cobalaminbinding protein activity is preferably TCII-R activity. The cytokine ispreferably an interferon such as interferon-β.

Another embodiment of the present invention is a composition that iscomprised of a metallocorrinoid and a cytokine. It is preferable thatthe metallocorrinoid be a vitamin B₁₂ analog, homolog, derivative orsimply vitamin B₁₂. This is particularly useful when there is adeficiency in vitamin B₁₂ or if the vitamin B₁₂ analog includes a drugconjugated thereto. It is particularly preferable that the vitamin B₁₂analog be a nitrosylcobalamin, but it may also be others known in theart, (e.g. hydroxocobalamin, cyanocobalamin, and methylcobalamin and 5′deoxyadenocobalamin or radiolabelled cobalamin derivatives). Thecomposition in accordance with this embodiment of the invention may alsoinclude a pharmaceutical carrier. It is preferable that the cytokine bean interferon, and more particularly interferon-β.

Another embodiment of the present invention is a therapeutic compositioncomprising a cobalamin or a cobalamin drug conjugate and a cytokine suchas interferon-β. In this embodiment, the therapeutic composition mayalso further comprise a pharmaceutical carrier. This is a particularadvantageous embodiment when the cobalamin drug conjugate is designedfor a specific aim in mind. Nitrosylcobalamin is just one cobalamin drugconjugate, and other drug conjugates may be selected from the groupconsisting of hydroxocobalamin, cyanocobalamin, methylcobalamin, and 5′deoxyadenocobalamin, radiolabelled cobalamin, or other cobalamin anddrug conjugate. This embodiment is particular useful in the treatment ofdiseases where the delivery of a therapeutic agent via a cobalamindelivery mechanism would be beneficial.

Another embodiment of the present invention is a method of enhancinguptake or activity of a metallocorrinoid comprised of administering acytokine. It is preferable that the metallocorrinoid be a vitamin B₁₂ ora vitamin B₁₂ analog, homolog, or derivative. In this method it ispreferable that the cytokine is an interferon, and more preferably thatthe interferon be interferon-β.

Another embodiment of the present invention is a method of enhancingcellular uptake of a metallocorrinoid comprising the step of contactinga cell with a cytokine, particularly where the step of contacting a cellwith a cytokine occurs through induction of cytokine. In thisembodiment, it is preferable that the metallocorrinoid is vitamin B₁₂ ora vitamin B₁₂ analog. As in other embodiments, the vitamin B₁₂ analogmay be any suitable vitamin B₁₂ analog, homolog or derivatives such as acobalamin drug conjugate. In this embodiment it is preferable that thecytokine is an interferon, particularly interferon-β.

Another embodiment of the present invention is a method of treating apatient comprising the steps of inducing cytokine production; andadministering a metallocorrinoid. The step of inducing cytokineproduction may include administering a cytokine, or administering anagent as is known in the art to stimulate cytokine expression orproduction. The metallocorrinoid of this embodiment may be vitamin B₁₂or a vitamin B₁₂ analog, homolog or derivative such as a cobalamin drugconjugate. The cytokine is preferably an interferon, more preferablyinterferon-β.

Yet another embodiment of the present invention is a method of enhancingbio-availability of a metallocorrinoid, comprising the step ofadministering interferon-βalone or in combination with ametallocorrinoid.

Yet another embodiment of the present invention is a method of treatinga subject to increase TCII-R activity in a cell comprising the step ofadministering to a subject in need of such treatment a cytokine toincrease TCII-R activity in an amount effective to increase TCII-Ractivity in said cell. In this embodiment, it is preferable that thesubject be cobalamin deficient. Another application of this embodimentis wherein the amount is sufficient to increase TCII-R activity abovenormal baseline levels. Preferably, this method may also be useful whenthe subject has an abnormally low level of TCII-R activity. This methodpreferably includes the step of co-administering a substrate (or ligand)of TCII-R, wherein the substrate of TCII-R is a cobalamin based compound(e.g. cobalamin or a cobalamin drug conjugate). The cobalamin drugconjugate is preferably nitrosylcobalamin, but may be any suitablecobalamin drug conjugate such as those known in the art.

Yet another embodiment of the present invention is a method of treatingcancer comprised of administering a cytokine (e.g. interferon-β) toenhance the uptake or increase the availability of cobalamin analogs,homologs, or derivatives. This can be done either alone or incombination with the cobalamin analog, homolog, or derivative.

Another embodiment of the present invention is a method of imagingtissue or cells through enhanced uptake of radiolabelled vitamin B₁₂analogs, homologs or derivatives via administration of a cytokine suchas interferon β.

Additional aspects and applications of the present invention will becomeapparent to the skilled artisan upon consideration of the detaileddescription of the invention, which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1 is a bar graph illustrating the anti-proliferative effect of acytokine (i.e. an interferon) and NO-Cbl on NIH-OVCAR-3 ovariancarcinoma.

FIG. 2 is a graph illustrating a median effect analysis in accordancewith the present invention.

FIG. 3 is a western blot analysis performed on extracts from ovariancarcinomas according to the present invention.

FIG. 4 illustrates a bar graph of a flow cytometric analysis of AnnexinV positive cells.

FIG. 5 are stained cells illustrating up-regulated TCII-R in control andIFN-βin NIH-OVCAR-3 treated samples.

FIG. 6 is a bar graph illustrating the anti-proliferative effect of acytokine (i.e. interferon) and NO-Cbl on WM9 melanoma.

FIG. 7 is a graph illustrating a median effect analysis on WM9 humanmelanoma cells.

FIG. 8 depicts treated and untreated WM9 tumor cells in accordance withthe present invention.

DETAILED DESCRIPTION OF THE INVENTION

While not wishing to be bound by theory, it appears that the uptake ofmetallocorrinoids such as vitamin B₁₂, NO-Cbl or other vitamin B₁₂-basedcompounds, is dependent upon the TCII receptor, specific for vitaminB₁₂. Because the TCII-R plays a central role in determiningmetallocorrinoid activity, the relationship between TCII-R and cytokines(e.g. interferons (“IFNs”)) was evaluated. IFNs upregulate theexpression of cell surface markers HLA-I, HLA-II, β2 microglobulin, andtumor associated antigens such as CEA and CA 125.

The present invention provides for an increase in receptor or receptoractivity responsible for the uptake of vitamin B₁₂ derived compounds.The administration of cytokines, particularly interferons such as IFN-βappears to enhance the activity of TCII-R. Administering these cytokinesprior to or concurrently with vitamin B₁₂-based compounds increases thedelivery of the vitamin B₁₂-based compounds and like metallocorrinoids.

Increased activity (e.g. TCII-R activity) can be accomplished in anumber of different ways. For example, an increase in the amount ofprotein or an increase in the activity of the protein (while maintaininga constant level of the protein) can result in increased “activity”. Anincrease in the amount of protein available can result from increasedtranscription of the gene, increased stability of the mRNA or a decreasein protein degradation.

The present invention, by causing an increase in Cbl-binding (e.g.TCII-R) activity, permits not only the re-establishment of normalbase-line levels of Cbl-binding activity, but also allows increasingsuch activity above normal base-line levels. Normal base-line levels arethe amounts of activity in a normal control group, controlled for ageand having no symptoms that would indicate alteration of Cbl-bindingactivity. The actual base line level will depend upon the particular agegroup selected and the particular measure employed to assay. When usingthe cytokines of the present invention not only can normal base-linelevels be restored, but abnormal activity can also be increaseddesirably far above normal base-line levels of TCII-R binding activity.Thus, “increasing activity” means any increase in Cbl-binding protein orcobalamin uptake in the subject resulting from the treatment, accordingto the invention, including, but not limited to, such activity as wouldbe sufficient to restore normal base-line levels, and such activity aswould be sufficient to elevate the activity above normal base-linelevels.

In one embodiment of the invention the increase in activity of theCbl-binding activity is cytokine induced. Cytokines are solublepolypeptides produced by a wide variety of cells. Cytokines control geneactivation and cell surface molecule expression. In what follows, theterm “cytokine” incorporates families of endogenous molecules of variousdenominations: lymphokines, monokines, interleukins, interferons,colonization factors and growth factors and peptides. The knowncytokines are in particular interferon-α (IFN-α.), interferon-β (IFN-β),γ-interferon (γ-IFN), interleukin-1 (IL-1) in α and β forms,interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4),interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-10 (IL-10),interleukin-12 (IL-12), tumor necrosis factor (TNF) in α and β forms,transforming growth factors (TGF-β), in β1, β2, β3, β1.2 forms, andcolony-stimulating factors (CSF) such as the granulocytemacrophage-stimulating factor (GM-CSF), the granulocytecolony-stimulating factor (G-CSF) and the macrophage-stimulating factor(M-CSF) and the epithelial growth factor (EGF), somatostatin,endorphins, the various “releasing factors” or “inhibitory factors” suchas TRF. There also exist pegilated forms of interferon. Cytokines playan essential role in the development of the immune system and thus inthe development of an immune response. However, besides their numerousbeneficial properties, they have also been implicated in the mechanismsfor the development of a variety of inflammatory diseases. For example,the cytokines TNF-α and IL-1 are thought to be part of the diseasecausing mechanism of atherosclerosis, transplant arteriosclerosis,rheumatoid arthritis, lupus, scleroderma, emphysema, etc.

Important embodiments of the invention involve populations never beforetreated with a cytokine such as interferon. Thus, the inventioninvolves, in certain aspects, treatments of individuals who areotherwise free of symptoms calling for treatment with interferons.

The cytokines and/or cobalamin compounds are preferably administered ineffective amounts. In general, an effective amount is any amount thatcan cause an increase in Cbl-binding proteins activity in a desired cellpopulation or tissue, and preferably in an amount sufficient to cause afavorable phenotypic change in the condition such as a lessening,alleviation or elimination of a symptom or of a condition.

With regard to the cobalamin or vitamin B₁₂ derived compounds, aneffective amount is that amount of a preparation that alone, or togetherwith further doses, produces the desired response. This may involve onlyslowing the progression of the disease temporarily, although morepreferably, it involves halting the progression of the diseasepermanently or delaying the onset of or preventing the disease orcondition from occurring. This can be monitored by routine methods.Generally, doses of active compounds would be from about 0.01 mg/kg perday to 1000 mg/kg per day. It is expected that doses ranging from 50-500mg/kg will be suitable, preferably intravenously, intramuscularly, orintradermally, and in one or several administrations per day.

Such amounts will depend, of course, on the particular condition beingtreated, the severity of the condition and the individual patientparameters. Some parameters for consideration include age, physicalcondition, size and weight, the duration of the treatment, the nature ofconcurrent therapy (if any), the specific route of administration andlike factors within the knowledge and expertise of the healthpractitioner. Intravenous administration and intramuscularadministration avoids transport problems associated with cobalamin whenadministered orally. However, if the vitamin B₁₂ analog, homolog orderivative is encapsulated, oral delivery may be preferred. In the eventthat a response in a subject is insufficient at the initial dosesapplied, higher doses (or effectively higher doses by a different, morelocalized delivery route) may be employed to the extent that patienttolerance permits. Multiple doses per day are contemplated to achieveappropriate systemic levels of compounds. It is preferred generally thata maximum dose be used, that is, the highest safe dose according tosound medical judgment. Those of ordinary skill in the art willunderstand, however, that a patient may insist upon a lower does ortolerable does for medical reasons, psychological reasons or forvirtually any other reason.

The cytokines (e.g, interferons) useful according to the invention maybe combined, optionally, with a pharmaceutically-acceptable carrier. Theterm “pharmaceutically-acceptable carrier” as used herein means one ormore compatible solid or liquid fillers, diluents or encapsulatingsubstances which are suitable for administration into a human. The term“carrier” denotes an organic or inorganic ingredient, natural orsynthetic, with which the active ingredient is combined to facilitatethe application. The components of the pharmaceutical compositions alsoare capable of being co-mingled with the molecules of the presentinvention, and with each other, in a manner such that there is nointeraction which would substantially impair the desired pharmaceuticalefficacy.

The pharmaceutical compositions may contain suitable buffering agents,including: acetic acid in a salt; citric acid in a salt; boric acid in asalt; and phosphoric acid in a salt. The pharmaceutical compositionsalso may contain, optionally, suitable preservatives, such as:benzalkonium chloride, chlorobutanol, parabens and thimerosal.

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular drug selected, theseverity of the condition being treated and the dosage required fortherapeutic efficacy. The methods of the invention, generally speaking,may be practiced using any mode of administration that is medicallyacceptable, meaning any mode that produces effective levels of theactive compounds without causing clinically unacceptable adverseeffects. Such modes of administration include oral, rectal, topical,nasal, intradermal, inhalation, intra-peritoneal, or parenteral routes.The term “parenteral” includes subcutaneous, intravenous, intramuscular,or infusion. Intravenous or intramuscular routes are particularlysuitable for purposes of the present invention.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well known in theart of pharmacy. All methods include the step of bringing the activeagent into association with a carrier that constitutes one or moreaccessory ingredients. In general, the compositions are prepared byuniformly and intimately bringing the active compound into associationwith a liquid carrier, a finely divided solid carrier, or both, andthen, if necessary, shaping the product.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the cytokines and/orcobalamins, which is preferably isotonic with the blood of therecipient. This aqueous preparation may be formulated according to knownmethods using suitable dispersing or wetting agents and suspendingagents. The sterile injectable preparation also may be a sterileinjectable solution or suspension in a non-toxic parenterally-acceptablediluent or solvent, for example, as a solution in 1,3-butane diol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono-or di-glycerides. In addition, fatty acids suchas oleic acid may be used in the preparation of injectables. Carrierformulation suitable for oral, subcutaneous, intravenous, intramuscular,etc. administrations can be found in Remington's Pharmaceutical Science,Mack Publishing Co., Easton, Pa. which is incorporated herein in itsentirety by reference thereto.

Other delivery systems can include time-released, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the active compound, increasing convenience to thesubject and the physician, and may be particularly suitable for certaincobalamin drug conjugates of the present invention, particularly thenitrosylcobalamin due to its activation under acidic conditions found inthe early gastrointestinal tract. Many types of release delivery systemsare available and known to those of ordinary skill in the art. Theyinclude polymer base systems such as poly(lactide-glycolide),copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters,polyhydroxybutyric acid, and polyanhydrides. Microcapsules of theforegoing polymers contains drugs are described in, for example, U.S.Pat. No. 5,075,109. Delivery systems also include non-polymer systemsthat are: lipids including sterols such as cholesterol, cholesterolesters and fatty acids or neutral fats such as mono- di- andtri-glycerides; hydrogel release systems; sylastic systems; peptidebased systems; wax coatings; compressed tablets using conventionalbinders and excipients; partially fused implants; and the like. Specificexamples include, but are not limited to: (a) erosional systems in whichthe active compound is contained in a form within a matrix such as thosedescribed in U.S. Pat. Nos. 4,452,775, 4,667,014, 4,748,034 and5,239,660 and (b) diffusional systems in which an active componentpermeates at a controlled rate from a polymer such as described in U.S.Pat. Nos. 3,832,253, and 3,854,480. In addition, pump-based hardwaredelivery systems can be used, some of which are adapted forimplantation.

Use of a long-term sustained release implant may be desirable. Long-termrelease, are used herein, means that the implant is constructed andarranged to delivery therapeutic levels of the active ingredient for atleast 30 days, and preferably 60 days. Long-term sustained releaseimplants are well-known to those of ordinary skill in the art andinclude some of the release systems described above.

In one aspect of the invention, the cytokine is “co-administered” with ametallocorrinoid which means administered substantially simultaneouslywith a metallocorrinoid. By substantially simultaneously, it is meantthat the cytokine (e.g. interferon-β) is administered to the subjectclose enough in time with the administration of the other agent (e.g.,vitamin B₁₂ or a cobalamin conjugate), whereby the two compounds mayexert an additive or even synergistic effect.

The following is provided as an illustration of the present invention asit applies to both in vivo and in vitro. The materials, methods,examples, results, and discussions should in no way be viewed as alimitation thereto. For simplicity, the materials and methods sectionsis provided after the following detailed discussion of the presentinvention.

Examples, Results and Discussion:

FIG. 1 illustrates NIH-OVCAR-3 ovarian carcinoma evaluated in accordancewith the present invention after 72-hrs growth. Cytokines, particularlyinterferons, appear to enhance the activity or upregulate the cellularreceptor for vitamin B₁₂ (TCII-R), resulting in enhanced TCII-R activity(in this case demonstrated by NO-Cbl uptake). Single agent andcombination drug effects were assessed to determine whether IFN-βenhanced NO-Cbl activity. As shown in FIG. 1, NIH-OVCAR-3 cells weretreated continuously with varying concentrations of NO-Cbl and IFN-β.Consistent with our hypothesis, we observed synergisticanti-proliferative activity between IFN-β and NO-Cbl. These matters areshown in the median effect analysis shown in FIG. 2 (similar toisobologram analysis) indicated synergy (a combination index <1) betweenNO-Cbl and IFN-β at all 3 doses tested. Cytotoxicity was noted at thehighest combination dose.

To assess the effect of IFNβ on TCII-R expression, a western blotanalysis was performed on extracts from NIH-OVCAR-3 cells (ovariancarcinoma) as shown in FIG. 3. Lane 1 is untreated. Lanes 2 and 3 areIFN β treated (200 u/ml) at 4 and 16 hrs respectively. Lanes 4 and 5 areliver and kidney extracts respectively, and serve as a positive control,since TCII-R is abundant in these tissues. As shown in FIG. 3, IFN βcauses an increase in the expression of the TCII receptor, identified asthe monomer at 62 kDa with the corresponding dimer at 124 kDa,consistent with TCII-R. These results correlate with theanti-proliferative effect of co-treatment of NIH-OVCAR-3 cells with IFNβ and nitrosycobalamin shown in FIG. 1. The increased expression of theTCII receptor by IFN β treatment results in the increased uptake ofnitrosylcobalamin and thus enhanced destruction of the cells. Theco-delivery of IFN-β and nitrosylcobalamin appears to result insynergistic destruction of tumor cells as a result of increased TCIIreceptor expression or activity.

A flow cytometric analysis of Annexin V positive cells was performed toassess the % apoptosis (programmed cell death) of NIH-OVCAR-3 cellstreated with NO-Cbl, alone and in combination with IFN-β. This isillustrated in FIG. 4. The ID₂₅ was used for both NO-Cbl (10 uM) andIFN-β (20 U/mL) for 48 hrs. The effect of IL-2 (250 U/mL) wereprotective against the effects of NO-Cbl.

To further elucidate IFN-β upregulated TCII-R, human NIH-OVCAR-3 tumorswere grown in nude mice to a size of 3 mm in diameter. The control groupreceived PBS and the treated group received human IFN-β 10⁵ units dailyfor three days. Tumors were harvested, paraffin embedded, and sectionswere stained with rabbit polyclonal anti-TCII-R antibody, (provided byDr. Seetharam's lab, Medical College of Wisconsin). FIG. 5 depicts thesetreatments. The left panel is an untreated tumor whereas the right panelis a tumor from a mouse that received IFN-β. The areas stained brownrepresent TCII-R. A comparison of the panels demonstrates increasedexpression of TCII-R with IFN β treatment. The increased expression ofthe TCII receptor allows for increased uptake of NO-Cbl, consistent withthe synergy observed in the SRB and Annexin V assays upon NO-Cblco-treatment with IFN-β.

WM9 human melanoma was evaluated after 4 days growth. This is shown inFIG. 6. WM9 cells were treated continuously with varying concentrationsof NO-Cbl and IFN-β. Similar to the NIH-OVCAR-3 cells, there wassynergistic anti-proliferative activity between IFN-β and NO-Cbl, as isshown in FIG. 7. Median effect analysis indicated synergy (a combinationindex <1) between NO-Cbl and IFN-β at all 3 doses tested.

To further elucidate whether IFN-β upregulated TCII-R, human WM9 tumorswere grown in nude mice to a size of3 mm in diameter. The control groupreceived PBS and the treated group received human IFN-β 10⁵ units dailyfor three days. Tumors were harvested, paraffin embedded, and sectionswere stained with rabbit polyclonal anti-TCII-R antibody, (provided byDr. Seetharam's lab, Medical College of Wisconsin). FIG. 8 depicts thesetreatments. The upper two panels are untreated tumors whereas the lowerpanels are tumors from mice that received IFN-β. The areas stained brownrepresent TCII-R. A comparison of the panels demonstrates increasedexpression of TCII-R with IFN β treatment.

One can see from the basal TCIIr activity in the NIH-OVCAR-3 and WM9stained sections that when TCII-R expression is lower, NO-Cbl uptake isnot pronounced. This is reflected by a higher ID₅₀ associated with theWM9 cells compared to NIH-OVCAR-3 tumors. Although interferonadministration in both NIH-OVCAR-3 and WM9 resulted in increasedeffectiveness of NO-Cbl, lower basal TCIIR expression in WM9 rendersthese cells less sensitive to the effect of NO-Cbl and the combinationwith IFN-β.

TCII-R is an important component of metallocorrinoid (e.g. vitamin B₁₂)metabolism and represents a site-specific target to regulate vitamin B₁₂uptake. Nitrosylcobalamin, a vitamin B₁₂ based carrier of nitric oxide(NO), was used to validate the in vivo functional relevance of increasedTCII-R expression. Intraperitoneal NO-Cbl treatment of establishedsubcutaneous NIH-OVCAR-3 tumors resulted in tumor regression. The meanvolume of untreated tumors was 18 fold greater compared to NO-Cbltreated tumors at the end of the study. Treated tumors decreased 4-foldin volume during the treatment period. There was no histologic evidenceof toxicity to normal tissues at NO-Cbl doses of 170 mg/kg/day after 60days. IFN-β treatment of NIH-OVCAR-3 cells in culture resulted inincreased expression of the TCII-R, detected as a monomer (62 kDa) and adimer (124 kDa). Similarly, immunohistochemical analysis of NIH-OVCAR-3xenografts from nude mice that received human IFN-β showed increasedTCII-R expression compared to controls. Tumors that were resistant toIFN-β and NO-Cbl in vivo exhibited minimal to no immunohistochemicalevidence of TCII-R upregulation. In culture, combination treatment withIFN-β and No-Cbl resulted in synergistic anti-proliferative activity inNIH-OVCAR-3 cells and several different human cells lines includingMCF-7 (breast), DU145 and LNCap (prostate), ACHN (renal), A549 (lung),WM9, WM35, WM164, and WM3211 (melanoma). Treatment of NIH-OVCAR-3 cellswith the combination of NO-Cbl and IFN-β resulted in a 2-fold increasein annexin V positive cells compared to NO-Cbl alone. Interestingly, aRibonucleotide Protection Assay revealed a ten-fold increase in TRAILand Caspase 7 in NIH-OVCAR-3 cells treated with the combination ofNO-Cbl and IFN-β. Therefore, up-regulation and/or increased activity ofthe TCII-R by IFN-β results in synergistic anti-tumor effects in vitroand in vivo.

Materials and Methods:

In-vivo IFN-β treatment of nude mice inoculated with tumors (e.g.WM9—human melanoma or NIH-OVCAR-3—ovarian carcinoma) andImmunohistochemical analysis: Nude mice (n=2 each group), wereinoculated with tumors (e.g. WM9—human melanoma or NIH-OVCAR-3—ovariancarcinoma), subcutaneously (s.c.), one tumor on each flank. The tumorswere grown until 3-5 mm in diameter. Human IFN-β (10⁵ units) wasadministered s.c. for three days to the treatment animals. On day four,animals were sacrificed and tumors were fixed in formalin and paraffinembedded. The sections were analyzed using standard immunohistochemicaltechniques. Anti-TCII-R was used as the primary antibody.

SRB Anti-Proliferative Cell Survival Assay:

Cells (2×10³) were seeded in 96-well plates. Data points represent meanof eight replicates. (n=8). A control plate was fixed 4 hr after seeding(to allow cells to attach) to determine the initial seeding density(A_(ini)). This was defined as 0% growth. To the wells of the seededexperimental plate, IFN-β was added and incubation continued for 3-5days. Untreated cell controls were included. Growth obtained with thiscontrol was defined as 100% (A_(fin)). To determine cell number, cellswere fixed with 10% trichloroacetic acid at 4° C. for 1 h. They werestained with 0.4% sulforhodamine B prepared in 1% acetic acid at 25° C.for 1 h (27). The wells were washed with 1% acetic acid. Bound dye waseluted with 100 μl of 10 mM Tris-HCl, pH 10.5 and quantitated in amicroplate reader at 570 nm. Growth in IFN-β treated wells(experimental=exp) was expressed as a percentage of untreated controlgrowth (mean±SEM).

% Control growth=100%×(A _(exp) −A _(ini))/(A _(fin) −A _(ini))

% STD=100%×(STD_(exp)/(A _(fin) −A _(ini)))

% SEM=100%×(SEM_(exp)/(A _(fin) −A _(ini))) where SEM=STD/n

Western Blot TCII Receptor:

Cells in culture were treated with vehicle (untreated) or with IFN-β(500 U/ml) for 4 and 16 hrs, washed twice in PBS, harvested by scraping,and lysed in buffer containing 100 mM saline-TRIS. Total cell extractswere homogenized prior to loading. Protein amounts in clarified cellextracts were determined using Bio-Rad protein assay reagent. Equivalentamounts of protein (100 μg) were loaded on 10% polyarcylamide SDSseparating gels and electrophoresis was performed using glycine-SDSbuffer. Following electrophoresis, gels were equilibrated in transferbuffer 30 min at 25° C., and proteins transferred to nitrocellulosemembrane.

Immunoblot With Electro-Chemiluminescense Detection:

All steps were performed at 25° C., Following 90 min electrophoretic wettransfer, the membranes were incubated in washing buffer TBS-Tween(1×TBS, 0.2% X-100,)+4% BSA for 1-2 hr to block non-specific binding.The membrane was washed in washing buffer. Membranes were then incubatedin 25 ml of primary antibody at 1:500 dilution in the washing bufferovernight at 4° C. Membranes were then washed using the washing bufferfour times, 10 min each. Membranes were incubated in 50 ml horseradishperoxidase-conjugated secondary antibody (Zymed) at 1:10,000 dilution inwashing buffer for 30 minutes. Membranes were washed in the washingbuffer for two hours. Equal volumes of electro-chemiluminescense (ECL)reagents A and B (Amersham) were mixed to give enough reagents todevelop the blot (0.125 ml/cm²), Excess buffer was drained from themembrane and it was placed protein side up on plastic wrap. Detectionreagent was added to the protein side of the membrane. The reaction wasallowed to continue for exactly 1 minute. Excess detection reagent wasdrained and the membrane was placed protein side down on plastic wrapand exposed to film for empirically determined lengths of time.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecomposition, methods, and in the steps or in the sequence of steps ofthe method described herein, without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

McLean G R, William M J, Woodhouse C S, Ziltener H J: Transcobalamin IIand in vitro proliferation of leukemic cells. Leuk Lymphoma 30: 101-9,1998.

Tsao C S, Myashita K: Influence of cobalamin on the survival of micebearing ascites tumor. Pathobiology 61: 104-8, 1993.

Jensen H S, Gimsing P, Pedersen F, Hippe E: Transcobalamin II as anindicator of activity in metastatic renal adenocarcinoma. Cancer 52:1700-4, 1983.

Tsao C S, Miyashita K, Young Cytotoxic activity of cobalamin in culturedmalignant and nonmalignant cells, Pathobiology 58: 292-6, 1990.

Shimizu N, Hamazoe R, Kanayama H, Maeta M, Koga S: Experimental study ofantitumor effect of methyl-B12. Oncology 44: 169-73, 1987.

McLean G R, Pathare P M, Wilbur D S, Morgan A C, Woodhouse C S, SchraderH W, Ziltener H J: Cobalamin analogues modulate the growth of leukemiacells in vitro. Cancer Res 57: 4015-22, 1997.

Huennekens F M, DiGirolamo P M, Fujii K. Jacobsen D W, Vitols K S:B12—dependent methionine synthetase as a potential target for cancerchemotherapy. Adv Enzyme Regul 14: 187-205, 1976.

Bauer, Joseph A., Characterization and nitric oxide release profile ofnitrosylcobalamin: a potential chemotherapeutic agent. Anti-Cancer Drugs1998; 9(3): 239-244.

1. A therapeutic composition comprising a metallocorrinoid and acytokine.
 2. The therapeutic composition of claim 1, wherein saidmetallocorrinoid is vitamin B₁₂.
 3. The therapeutic composition of claim1, wherein said metallocorrinoid is a vitamin B₁₂ analog.
 4. Thecomposition of claim 1, wherein said cytokine is interferon-β.
 5. Thecomposition of claim 3, wherein said vitamin B₁₂ analog isnitrosylcobolamin.
 6. The therapeutic composition of claim 3, whereinsaid vitamin B₁₂ analog is selected from the group consisting ofhydroxocobalamin, cyanocobalamin, methylcobalamin, 5′deoxyadenocobalamin.
 7. The therapeutic compsition of claim 1 whereinsaid metallocorrinoid is a cobalamin drug conjugate and said cytokine isinterferon-β.
 8. The therapeutic composition of claim 7, furtherincluding a pharmaceutical carrier.
 9. A method of enhancing uptake of ametallocorrinoid comprised of administering a cytokine.
 10. The methodof claim 9, wherein said metallocorrinoid is vitamin B₁₂.
 11. The methodof claim 9, wherein said metallocorrinoid is a vitamin B₁₂ analog. 12.The method of claim 9, wherein said cytokine is an interferon.
 13. Themethod of claim 12, wherein said interferon is interferon-β.
 14. Amethod of treating a patient comprising the steps of inducing cytokineproduction and administering a metallocorrinoid.
 15. The method of claim14, where the step of inducing cytokine production comprisesadministering a cytokine.
 16. The method of claim 14, wherein saidmetallocorrinoid is vitamin B₁₂.
 17. The method of claim 14 wherein saidmetallocorrinoid is a vitamin B₁₂ analog.
 18. The method of claim 14,wherein the cytokine is interferon-β.
 19. A method for increasing TCII-Ractivity in a subject to treat a condition favorably affected by anincrease in said TCII-R activity comprising the step of administering toa subject in need of such treatment a cytokine in an amount effective toincrease TCII-R activity in the subject.
 20. The method of claim 19,further comprising the step of co-administering a vitamin B₁₂ analog.21. The method of claim 19, wherein said cytokine is administered priorto the vitamin B₁₂ analog.
 22. The method of claim 19, wherein saidcytokine is administered prophylactically.
 23. The method of claim 19,wherein said cytokine is administered acutely.
 24. The method of claim19, wherein said cytokine is an interferon.
 25. The method of claim 24,wherein said interferon is interferon-β.
 26. The method of claim 19,wherein said condition is unwanted cellular proliferation.
 27. A methodof enhancing bio-availability of a metallocorrinoid comprising the stepof administering interferon-β.
 28. A method of treating a subject toincrease TCII-R activity in a cell comprising the step of administeringto a subject in need of such treatment a cytokine in an amount effectiveto increase TCII-R activity in said cell.
 29. The method of claim 28,wherein the subject is cobalamin deficient.
 30. The method of claim 28,wherein the amount is sufficient to increase TCII-R activity abovenormal baseline levels.
 31. The method of claim 28, wherein the subjecthas an abnormally low level of TCII-R activity.
 32. The method of claim28, further comprising the step of co-administering a ligand of TCII-R.33. The method of claim 32 herein the ligand of TCII-R is cobalamin. 34.The method of claim 32, wherein the ligand of TCII-R is a cobalamin drugconjugate.
 35. The method of claim 34, wherein the cobalamin drugconjugate is nitrosylcobalamin.